Biological mode of action of a nitrophenolates-based ... · Przybysz et al. Mode of action of a nitrophenolates-based biostimulant (0.1%), and water. Atonik has been used successfully

ORIGINAL RESEARCH ARTICLEpublished: 16 December 2014doi: 10.3389/fpls.2014.00713

Biological mode of action of a nitrophenolates-basedbiostimulant: case studyArkadiusz Przybysz1*, Helena Gawronska1 and Janina Gajc-Wolska2

1 Laboratory of Basic Research in Horticulture, Faculty of Horticulture, Biotechnology and Landscape Architecture, Warsaw University of Life Sciences – SGGW,Warsaw, Poland

2 Department of Vegetable and Medicinal Plants, Faculty of Horticulture, Biotechnology and Landscape Architecture, Warsaw University of Life Sciences – SGGW,Warsaw, Poland

Edited by:

Ebrahim Hadavi, Karaj Branch,Islamic Azad University, Iran

Reviewed by:

Bernard Dumas, Centre National dela Recherche Scientifique, FranceMarcin Kozak, UniwersytetPrzyrodniczy we Wroclawiu, PolandYoury Pii, Free University of Bolzano,Italy

*Correspondence:

Arkadiusz Przybysz, Laboratory ofBasic Research in HorticultureFaculty of Horticulture,Biotechnology and LandscapeArchitecture, Warsaw University ofLife Sciences – SGGW,Nowoursynowska 159,Warsaw 02-776, Polande-mail: [email protected]

The challenges facing modern plant production involve (i) responding to the demand forfood and resources of plant origin from the world’s rapidly growing population, (ii) copingwith the negative impact of stressful conditions mainly due to anthropopressure, and (iii)meeting consumers’ new requirements and preferences for food that is high in nutritivevalue, natural, and free from harmful chemical additives. Despite employing the mostmodern plant cultivation technologies and the progress that has been made in breedingprograms, the genetically-determined crop potential is still far from being fully exploited.Consequently yield and quality are often reduced, making production less, both profitableand attractive. There is an increasing desire to reduce the chemical input in agricultureand there has been a change toward integrated plant management and sustainable,environmentally-friendly systems. Biostimulants are a category of relatively new productsof diverse formulations that positively affect a plant’s vital processes and whose impactis usually more evident under stressful conditions. In this paper, information is providedon the mode of action of a nitrophenolates-based biostimulant, Atonik, in model speciesand economically important crops grown under both field and controlled conditions ina growth chamber. The effects of Atonik on plant morphology, physiology, biochemistry(crops and model plant) and yield and yield parameters (crops) is demonstrated. Effectsof other biostimulants on studied in this work processes/parameters are also presented indiscussion.

Keywords: biomass accumulation, efficiency of photosynthetic apparatus, growth and development,

nitrophenolates, water status, yield, yield parameters

INTRODUCTIONThe challenge facing modern plant production nowadays is torespond to the increasing demand for food and resources ofplant origin by the world’s rapidly growing population. Yieldis negatively affected by various adverse environmental condi-tions and increasing anthropopression and despite employingthe most modern plant cultivation technologies and the progressbeing made in breeding programs, the genetically-determinedcrop potential is still far from being fully exploited. Accordingto Bray et al. (2000), stresses can reduce average productivityby 65–87%, depending on the crop. This consequently makesplant production less profitable for farmers and less attractive forconsumers.

Biostimulants syn. biostimulators are a category of relativelynew products of diverse formulations that positively affect aplant’s vital processes (Calvo et al., 2014), usually more evidentunder stressful conditions, by increasing a plant’s tolerance tostresses and repairing damage caused by unfavorable conditions.

Biostimulants may be of natural or synthetic origin and consistof various organic and inorganic components. Among natu-rally derived biostimulants are preparations based on free aminoacids, extracts from seaweed and fruit, effective microorganisms,

humic substances, and chitosan (Calvo et al., 2014). Syntheticbiostimulants are composed, among others, of plant growth reg-ulators, phenolic compounds, inorganic salts, essential elements,and other substances that have stimulating properties for plants.

Although the term “biostimulant” has been used for manyyears, it is still not fully defined. The European BiostimulantIndustry Council (EBIC) describes biostimulants as a prepara-tions “. . . containing substance(s) and/or micro-organisms whosefunction, when applied to plants or the rhizosphere is to stimu-late natural processes to enhance/benefit nutrient uptake, nutri-ent efficiency, tolerance to abiotic stress, and crop quality. . . .”Biostimulants do not replace, but rather complement plant pro-tection products and fertilizers. They have no direct action againstpests and they operate through different mechanisms than fertil-izers, regardless of the occasional presence of nutrients in theseproducts (http://www.biostimulants.eu).

It is impossible to suggest one common mode of action for allbiostimulants, therefore this work focused on Atonik, known asChapperone (USA) or Asahi SL (Poland). Atonik is a Japanesesynthetic biostimulant composed of three phenolic compounds:sodium para-nitrophenolate PNP (0.3%), sodium ortho-nitrophenolate ONP (0.2%) and sodium 5-nitroguaiacolate 5NG

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Przybysz et al. Mode of action of a nitrophenolates-based biostimulant

(0.1%), and water. Atonik has been used successfully for manyyears in the cultivation of most important crops worldwide. Itspositive effect on yield is already well proven (Djanaguiramanet al., 2004a, 2009; Bynum et al., 2007; Grajkowski and Ochmian,2007; Budzynski et al., 2008; Cerný et al., 2008; Kositornaand Smolinski, 2008; Kozak et al., 2008a; Malarz et al., 2008;Michalski et al., 2008; Sawicka and Mikos-Bielak, 2008), butknowledge about its mode of action has, until this study, beenfragmented, not covered thoroughly in literature, and sometimeseven controversial. Early works described some of the poten-tial positive properties of Atonik. It has been shown that thenitrophenolates making up this biostimulant increase cytoplasmstreaming (Yamaki et al., 1953; Wilson and Kaczmarek, 1993).Plants treated with nitrophenolates have greater inhibition of IAAoxidase, which ensures a higher activity of naturally synthesizedauxins (Stutte and Clark, 1990). The phosphorylated formof para-nitrophenolate enhances IAA activity when used as asubstrate for phosphatases via increased high-affinity bindingsites of IAA (Davies, 1987) and could be as effective as ATP(Koizumi et al., 1990). According to Stutte et al. (1987), plantsexposed to nitrophenolates uptake more nutrients from themedium. Furthermore, Sharma et al. (1984) showed a significantincrease in the activity of nitrate reductase, an important enzymein nitrogen metabolism.

More recent studies prove that Atonik positively affects variousprocesses controlling plant growth, development and productiv-ity. Biostimulant-treated plants are more advanced in growth anddevelopment (Djanaguiraman et al., 2005b; Gulluoglu et al., 2006;Kozak et al., 2008a; Borowski and Blamowski, 2009) and accumu-late more biomass (Gruszczyk and Berbec, 2004; Djanaguiramanet al., 2005a, 2009; Kołodziej, 2008). Atonik increases the intensityof photosynthesis (Borowski and Blamowski, 2009) and tran-spiration rate, but usually without a reduction in relative watercontent (Wróbel and Wozniak, 2008; Borowski and Blamowski,2009). The positive effects of Atonik are much more evident whenplants are grown under adverse conditions. It has been foundthat biostimulants play a protective role against various abioticstresses, such as low or high temperatures, drought, heavy met-als, and salinity (Gulluoglu et al., 2006; Gawronska et al., 2008;Wrochna et al., 2008; Borowski and Blamowski, 2009). Moreover,some results have indicated that if plants were grown under opti-mal conditions, the positive effect of this preparation might notbe recorded (Budzynski et al., 2008; Ksiezak, 2008).

However, the works presented above individually only covera narrow range of processes and/or parameters. This paper pro-vides the first comprehensive study of the Atonik mode of actionand demonstrates the effects of biostimulant on yield and its com-ponents, plant morphology, physiology and biochemistry in themodel plant Arabidopsis thaliana L. and some crops that are eco-nomically important (Brassica napus L. var. oleifera and Cucumissativus L.).

MATERIALS AND METHODSThe experiments were carried out on crops: oilseed rape andcucumber and A. thaliana used as model plant. Plants were grownin field conditions and growth chambers under optimal, droughtor noble metal stresses. Concentrations of Atonik and the number

of its applications were first determined in preliminary studies inorder to ensure a stimulative/protective effect of the biostimulantin particular species and growing conditions.

EFFECT OF ATONIK ON FIELD-GROWN OILSEED RAPE PLANTSPlant material and growing conditionsOilseed rape cv. “Lisek” plants were cultivated in the 2007 and2008 growing seasons in the experimental field in Chylice of theWarsaw University of Life Sciences—SGGW. The field is situated105 m above sea level and located at 22◦33′25′′ N and 52◦05′71′′E. The 30-year average annual temperature and rainfall are 7.8◦C(12.8◦C during the growing seasons) and 592 mm (448 mm dur-ing the growing seasons) respectively. The soil (black degraded,composed of loamy sand) is classified as average good, with a0.8–1.6% content of organic matter and pH 6.0–6.2. Experimentswere conducted in completely randomized blocks in four repli-cates (plots of 18 or 14.4 m2 in 2007 and 2008, respectively).The seeds were sown at a spacing of 30 × 6.5 cm. For the mea-surements, five plants from each plot were chosen. In the 2007growing season the experimental plants were grown in 25 L potsfilled with soil taken from the particular plots, and the pots wereplaced (buried) on the appropriate plots, following a statisti-cal design. Routine agricultural practices recommended for thisspecies and location were employed. Both vegetative seasons werecharacterized by similar growing conditions, the only exceptionwas a strong late spring frost in 2007. Atonik was applied in springas a single (BBCH 29–31) or double (BBCH 29–31 and 51) foliarspray in a concentration of 0.2% v/v in 300 L ha−1. NPK fertil-izers were applied as 194 kg N ha−1 (34—autumn, 160—spring),80 kg P ha−1 and 120 kg K ha−1.

Measured parameters/processesOne (2007) or three (2008) weeks after the Atonik application,the following parameters were measured: (i) plant gas exchange:intensity of photosynthesis and transpiration, stomatal resistance(Photosynthesis System LICOR 6200, Lincoln, NE, USA), (ii)chlorophyll content (CCM-200, OPTI-SCIENCES, USA) and (iii)chlorophyll a fluorescence (Handy PEA, Hansatech, UK). Themeasurements were performed for 9 (2007) and 10 (2008) weeks.After harvest, (i) the height of the plants was measured, (ii) thenumber of leaves, primary laterals, pods and seeds in pods werecounted, and (iii) the accumulation of biomass (after drying at105◦C for 2 h and then at 75◦C for 48 h) and yield of seeds (viaweighing of air dry seeds) were recorded.

EFFECT OF ATONIK ON FIELD-GROWN CUCUMBER PLANTSPlant material and growing conditionsCucumber cvs. “Octopus F1” (Syngenta Seeds), Opera F1 andSonate F1 (both Rijk Zwaan) plants were cultivated in the 2012growing season in the experimental field of the Department ofVegetable and Medicinal Plants at Wilanów, Poland. Plants weregrown in deep medium-heavy alluvial soil (classified as good)with a 1.9–2.3% content of organic matter and pH 6.0–6.5. Theexperiment was arranged in a two-factor split-plot design withfour replicates (plots of 6 m2). Seeds were sown manually on 14May into plastic pots of 8 cm diameter filled with peat substrate.On 24 May, when the plants had 1–2 leaves, seedlings were planted

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in the field at a spacing of 30 × 150 cm. There were 14 plants inthe plot. Atonik was applied as a foliar spray (12 and 27 June and27 July) in a concentration of 0.1% v/v in 500 L ha−1. Controlplants were treated with water. During the period of water short-age, plants were T-Tape irrigated. The soil content of N, P, andK was kept at the optimum level, with fertilizers applied to equalthe average of 150 kg N ha−1 (60 kg N side dressing), 50 kg P ha−1

and 190 kg K ha−1. The harvest was carried out successively, twicea week (13 times), starting from the middle of September.

Measured parameters/processesAt harvest, the total and marketable yield was recorded. Yieldquality was evaluated by determining the content of: (i) dry mat-ter (drying to constant weight at 105◦C), (ii) sugars (Luff–Schoorlmethod), (iii) vitamin C (titration with Tillmans’ method),(iv) nitrates (spectrophotometer Tecator Fiastar 5010 at wave-length 540 nm), (v) phosphorus (spectrophotometer Shimadzu1700 at wavelength 460 nm), (vi) potassium, and (vii) calcium(both using flame spectrophotometer Sherwood Model 410).Marketable fruits were graded according to the Polish standardPN-85/R-75359 into two pickling grades of (i) 6–10 cm long witha diameter of 2.5–4.5 cm and (ii) 9–15 cm long with a diameter of4.5–5.5 cm.

EFFECT OF ATONIK ON A. THALIANA PLANTS GROWN UNDEROPTIMAL, DROUGHT, AND Pt STRESSPlant material and growing conditionsA. thaliana Col 4 seeds (Lehle Seeds, Round Rock, TX, USA) weresown onto multiplates filled with substrate (Universal Kronenerdesoil and sand in the proportion 2:1 v/v). Uniform, 6-week-oldseedlings were transplanted to (i) pots (Ø 10 cm) containing thesame substrate or (ii) hydroponics culture filled with 0.3 dm3

of a Hoagland solution (Arnon and Hoagland, 1940) modifiedby Siedlecka and Krupa (2002). The nutrient solution was con-tinuously aerated and renewed weekly. Plants were grown ingrowth chambers (Simez Control s.r.o. Vsetin, Czech Republic)at 22/18◦C with a photoperiod 8/16 h day/night, irradiance of250–280 μmol m−2s−1 PAR and relative humidity of 60%.

Drought stressBefore drought treatment, the maximum water capacity (MWC)of the substrate was determined. Drought stress was imposed onthe soil as a result of a daily limited water supply via pot weighingto the levels of 50, 40, 30, and 20% of MWC (three experiments)or 45 and 25% of MWC (two experiments). Depending on theexperiment, the combination consisted of 6–12 plants. On theday on which the substrate attained the desired MWC, the plantswere treated once with Atonik as a foliar spray at a concentra-tion of 0.1% (with an amount of water equal to 300 L ha−1 in thefield conditions) and grown for a further 4 weeks. Control plantswere cultivated at 60 or 65% MWC (optimal water conditions)and sprayed with distilled water.

Pt stressDuring the first week the nutrient solution was used at halfstrength and thereafter the complete composition of macro- andmicroelements was supplied. Two weeks after plants were trans-planted to hydroponics, during the nutrient solution change,

Pt and Atonik were added. Pt, in oxidation state II, wasadded at concentrations of 2.5, 25, and 50 μM in the formof [Pt(NH3)4](NO3)2. Atonik was added at a concentration of0.005% v/v. After treatment, the plants were grown for a further3 weeks. In total, three experiments were carried out, with 5–6 plants per combination. Control plants were grown in Pt andAtonik-free medium.

Measured parameters/processesDuring plant growth the following parameters were measured:(i) plant gas exchange: intensity of photosynthesis and transpi-ration, stomatal resistance (Photosynthesis System LICOR 6200,Lincoln, NE, USA), (ii) chlorophyll content (CCM-200, OPTI-SCIENCES, USA), (iii) chlorophyll a fluorescence (Handy PEA,Hansatech, UK), and (iv) water uptake (via daily pot weigh-ing). At harvest, sub-samples were collected for (i) relative watercontent (RWC, via weighing) and (ii) membrane injury (conduc-tometrically, MultiLevel 1, WTW, Germany) and data recorded on(iii) the height of plants, (iv) length and number of inflorescences,(v) number of pods, (vi) number and area of leaves (Leaf Area,Root Length and Image Analyzing System, Skye, UK), and (vii)biomass accumulated by the whole plants and particular organs(after drying at 105◦C for 2 h and then at 75◦C for 48 h).

STATISTICSThe number of replications, depending on the parameter, wasbetween 3 and 36, and is indicated in the specific tables or fig-ures. Differences between the combinations were evaluated withone or two-factor analysis of variance by LSD (Student’s t-test) orHSD (Tukey test) at α = 0.05. The presented data are mean ± SE(where indicated).

RESULTSEFFECT OF ATONIK ON FIELD-GROWN OILSEED RAPE PLANTSAtonik-treated plants in the 2007 season were taller than the con-trol, and produced slightly more pods (0.1–4.1%) and seeds inpods (0.9–2.8%) (Table 1). On the other hand these plants devel-oped a lower number of primary laterals. In the 2008 season,the biostimulant had no effect on the plants’ height. Regardless

Table 1 | Effect of Atonik on selected morphological parameters of

oilseed rape plants.

Combination Height Number of (plant−1)

(cm plant−1)Laterals Pods Seeds

2007 GROWING SEASON

Control 108.90 (±1.38) 5.55 (±0.27) 100.45 (±5.50) 15.92 (±0.14)

Atonik 1× 118.89* (±1.81) 5.42 (±0.21) 104.58 (±5.17) 16.06 (±0.13)

Atonik 2× 111.28 (±1.43) 5.22 (±0.24) 100.56 (±7.27) 16.37 (±0.15)

2008 GROWING SEASON

Control 162.10 (±1.29) 10.05 (±0.25) 253.00 (±8.50) 25.46 (±0.18)

Atonik 1× 161.63 (±1.16) 11.31 (±0.26) 273.63 (±11.87) 25.98 (±0.42)

Atonik 2× 160.05 (±2.13) 11.00 (±0.24) 250.08 (±8.91) 25.65 (±0.71)

Presented data are Mean ± SE, n = 20.*Values differ significantly at α = 0.05 as determined by LSD of t-Student test.





of whether Atonik was used once or twice, the number of later-als (9.5–12.5%) and seeds (0.7–2%) was greater. Only the singlespray increased the number of pods (8.1%) (Table 1).

The fresh weight of Atonik-treated plants in 2007 was 12.5%higher than that of the control and in the case of dry mat-ter Atonik contributed to an increase of between 11.9–23.7%(Table 2). The fresh weight and dry matter of stem and pods withseeds were also greater. Higher values were obtained for a sin-gle spray. In the next season the positive influence of Atonik onaccumulated biomass was less evident and was recorded after asingle spray only. Atonik slightly increased the fresh weight anddry matter of the aboveground part, the main stem and pods withseeds. The weight of the laterals was adversely affected. The yieldof plants sprayed once with Atonik exceeded the control by 35%(2007) or by just 3.6% (2008). After the double application nopositive effect or even reduction was noted (Table 2).

In the 2007 season, irrespective of the number of treatments,Atonik increased photosynthesis intensity (1–22%) and this effectlasted up to 7 weeks following the first spray (Table 3). In thefollowing year the positive effect on this process remained for 4weeks (3.6–20.3%). In the 2007 season, the sprayed plants wereusually characterized by a higher intensity of transpiration andlower stomatal resistance. In contrast to this, in the 2008 sea-son the effect of Atonik on these parameters was ambiguous. Thetotal chlorophyll content in both growing seasons was, with a fewexceptions, higher in biostimulant-treated plants (Table 3).

Measurements of chlorophyll a fluorescence showed that inthe 2007 season Atonik did not affect Fv/Fm (maximum quan-tum efficiency of Photosystem II) and P.I. (Performance Index)up to the late spring frost (−4.2◦C) that occurred between the36 and 39th day after the first application of the biostimulant(Figure 1A). Following the frost, a lowering in the Fv/Fm and P.I.values in the control was recorded, while in the treated plants theydid not change. Moreover, the positive effect on P.I. remained for

the next 22 days. The values of these parameters in the 2008 sea-son during the 8 weeks after the first spray were similar betweenthe treated and untreated plants. Starting from week 10, a reduc-tion in these parameters after the application of Atonik was noted(Figure 1B).

EFFECT OF ATONIK ON FIELD-GROWN CUCUMBER PLANTSThere was no significant effect of the Atonik on total ormarketable yield, or any interactions of both traits examined(Table 4). Slightly increased yields after biostimulant treatmentwere recorded only for the cultivar Octopus F1. Yields of fruitswere significantly related to the cultivar. The highest values offruit mass were recorded for cultivar Sonate F1 and the lowestfor Octopus F1. On average, for all the examined cultivars, thecontent of dry matter and soluble solids were significantly higherafter treatment with the biostimulant. When the cultivars wereexamined separately, it came out that dry content increased byAtonik, except in Opera F1. Soluble solids were always higher inplants sprayed with the biostimulant. The content of nitrates washigher on average in plants treated with Atonik. The exceptionwas the Sonate F1 cultivar. In plants sprayed with the biostimu-lant, a higher content of phosphorus was recorded. The content ofpotassium was only significantly affected by the cultivar and thehighest was found in Octopus F1, while the lowest was in SonateF1. The content of calcium was affected by both the biostimu-lant and the cultivar. The effect of Atonik on this parameter wasadverse and the Sonate F1 cultivar was characterized as having thegreatest content of calcium and Opera F1 the lowest (Table 4).

EFFECT OF ATONIK ON A. THALIANA PLANTS GROWN UNDEROPTIMAL, DROUGHT, AND Pt STRESSESOptimal and drought conditionsAtonik had a positive effect on A. thaliana grown in optimal con-ditions and clearly diminished the negative impact of drought

Table 2 | Effect of Atonik on biomass accumulation and seed yield in oilseed rape plants.

Measured parameter Combination g plant −1

Whole plant Pods with seeds Main stem Laterals Yield

2007 GROWING SEASON

Fresh weight Control 37.42 (± 1.96) 14.00 (± 0.85) 19.69 (± 1.02) 3.74 (± 0.37)

Atonik 1× 42.10 (± 2.79) 16.15 (± 1.17) 22.36 (± 1.36) 3.59 (± 0.34)

Atonik 2× 42.18 (± 2.78) 15.94 (± 1.97) 21.21 (± 1.43) 5.02 (± 0.50)

Dry matter Control 9.60 (± 0.51) 4.33 (± 0.27) 4.21 (± 0.20) 1.03 (± 0.10) 2.29 (± 0.16)

Atonik 1× 11.87 (± 0.66) 5.52 (± 0.36) 5.33 (± 0.26) 1.02 (± 0.08) 3.09 (± 0.23)

Atonik 2× 10.75 (± 0.67) 4.60 (± 0.39) 4.93 (± 0.26) 1.21 (± 0.10) 2.32 (± 0.24)

2008 GROWING SEASON

Fresh weight Control 147.86 (± 6.24) 59.86 (± 2.60) 62.84 (± 2.55) 25.15 (± 1.71)

Atonik 1× 154.26 (± 5.68) 61.28 (± 2.59) 69.69 (± 2.32) 23.28 (± 1.31)

Atonik 2× 136.82 (± 5.44) 50.89 (± 2.01) 63.58 (± 2.32) 22.36 (± 1.58)

Dry matter Control 68.46 (± 2.50) 42.12 (± 1.55) 15.75 (± 0.53) 10.59 (± 0.58) 24.69 (± 0.94)

Atonik 1× 70.18 (± 2.19) 44.11 (± 1.44) 16.34 (± 0.37) 9.73 (± 0.46) 25.58 (± 1.03)

Atonik 2× 62.55 (± 2.42) 38.03 (± 1.55) 15.33 (± 0.45) 9.18 (± 0.48) 21.93 (± 0.93)

Presented data are Mean ± SE, n = 20.






Table 3 | Effect of Atonik on the intensity of photosynthesis and transpiration, stomatal conductance, transpiration and chlorophyll content in

oilseed rape plants.

Measured

parameter

Combination Days after spray

2007 growing season

7 15 25 32 39 46 54 61

Gro

win

gse

ason

2006

/07

Photosynthesis(μmol CO2

m−2 s−1)

Control 7.96 (± 0.14) 11.84 (± 0.16) 10.03 (± 0.16) 10.10 (± 0.15) 12.42 (± 0.32) 10.78 (± 0.25) 7.21 (± 0.24) 5.38 (± 0.12)

Atonik 1× 8.02 (± 0.12) 13.32* (± 0.22) 10.11 (± 0.15) 10.68 (± 0.21) 14.49* (± 0.34) 11.56 (± 0.30) 7.89 (± 0.28) 5.13 (± 0.12)

Atonik 2× − − 11.29* (± 0.24) 12.30* (± 0.31) 12.96 (± 0.27) 11.63 (± 0.32) 7.50 (± 0.31) 5.09 (± 0.14)

Stomatalresistance(s cm−1)

Control 4.60 (± 0.08) 2.83 (± 0.05) 2.52 (± 0.05) 1.59 (± 0.06) 0.69 (± 0.04) 0.99 (± 0.07) 1.93 (± 0.07) 2.17 (± 0.16)

Atonik 1× 4.45 (± 0.13) 2.19* (± 0.04) 3.23* (± 0.10) 1.69 (± 0.05) 0.61 (± 0.02) 1.02 (± 0.05) 3.07* (± 0.19) 1.88 (± 0.08)

Atonik 2× − − 2.35 (± 0.04) 1.58 (± 0.02) 0.67 (± 0.03) 0.89 (± 0.05) 2.27 (± 0.13) 1.55* (± 0.07)

Transpiration(μmol H2Om−2 s−1)

Control 1.17 (± 0.02) 2.97 (± 0.02) 2.37 (± 0.03) 2.48 (± 0.07) 5.35 (± 0.09) 6.93 (± 0.19) 6.29 (± 0.14) 7.68 (± 0.31)

Atonik 1× 1.51 (± 0.11) 3.56 (± 0.21) 2.12* (± 0.04) 2.15 (± 0.05) 6.39* (± 0.09) 7.14 (± 0.17) 6.08 (± 0.28) 7.09 (± 0.21)

Atonik 2× − − 2.51 (± 0.03) 2.66 (± 0.07) 6.04* (± 0.14) 7.62 (± 0.25) 6.63 (± 0.29) 8.03 (± 0.21)

Chlorophyllcontent

Control − − − − − − 29.95 (± 1.42) 45.18 (± 0.54)

Atonik 1× − − − − − − 31.37 (± 1.47) 48.33 (± 0.62)

Atonik 2× − − − − − − 29.97 (± 0.70) 47.62 (± 0.63)

2008 growing season

21 28 38 54 68

Gro

win

gse

ason

2007

/08

Photosynthesis (μmol CO2 m−2 s−1) Control 9.79 ( ± 0.27) 8.76 ( ± 0.22) 12.24 ( ± 0.24) 11.29 ( ± 0.24) 10.79 ( ± 0.23)

Atonik 1× 11.38* ( ± 0.32) 9.60 ( ± 0.18) 11.57 ( ± 0.19) 10.01* ( ± 0.17) 9.31* ( ± 0.20)

Atonik 2× 10.15 ( ± 0.25) 10.54* ( ± 0.24) 11.55 ( ± 0.26) 11.10 ( ± 0.24) 10.55 ( ± 0.25)

Stomatal resistance (s cm−1) Control 0.81 ( ± 0.02) 1.22 ( ± 0.05) 0.53 ( ± 0.04) 0.83 ( ± 0.05) 1.71 ( ± 0.07)

Atonik 1× 1.25* ( ± 0.04) 1.37 ( ± 0.05) 0.49 ( ± 0.04) 0.91 ( ± 0.06) 3.18* ( ± 0.23)

Atonik 2× 1.36* ( ± 0.07) 2.01* ( ± 0.11) 0.51 ( ± 0.04) 0.77 ( ± 0.04) 1.98 ( ± 0.11)

Transpiration (μmol H2O m−2 s−1) Control 4.80 ( ± 0.07) 4.56 ( ± 0.07) 6.77 ( ± 0.14) 8.02 ( ± 0.23) 9.05 ( ± 0.40)

Atonik 1× 4.40 ( ± 0.12) 3.98* ( ± 0.06) 8.05* ( ± 0.16) 8.89 ( ± 0.33) 7.09 ( ± 0.53)

Atonik 2× 3.99* ( ± 0.11) 3.69* ( ± 0.10) 7.25 ( ± 0.18) 8.74 ( ± 0.27) 8.95 ( ± 0.72)

Chlorophyll content Control 38.08 ( ± 1.11) 38.76 ( ± 0.98) 30.92 ( ± 0.89) 25.06 ( ± 0.50) 18.43 ( ± 0.35)

Atonik 1× 36.26 ( ± 0.90) 40.09 ( ± 0.81) 30.79 ( ± 0.80) 27.50 ( ± 0.64) 21.59* ( ± 0.61)

Atonik 2× 36.28 ( ± 0.85) 39.40 ( ± 0.89) 32.98 ( ± 0.87) 25.14 ( ± 0.62) 22.16* ( ± 0.54)

Presented data are Mean ± SE, n = 24 or 36 (chlorophyll content).*Values differ significantly at α = 0.05 as determined by LSD of t-Student test.

(Figure 2). Plants sprayed with Atonik were taller and devel-oped more inflorescences (by 14–56%) and pods (by 93–450%)(Table 5). In 20 and 30% of MWC their number reduced. Leafarea was always greater in Atonik-treated plants, and this increaseranged between 3–43% (Table 5).

A. thaliana treated with Atonik produced more biomass andthis was true for optimal conditions and every level of droughtstress (Table 6). The increase of biomass accumulation recordedranged between 2.5–46 and 1–47%, respectively for fresh weightand dry matter. The positive effect of Atonik was more evident inthe case of generative organs (Table 6).

The efficiency of the photosynthetic apparatus of A. thalianaplants was positively affected by the biostimulant (Table 7).The intensity of photosynthesis was usually higher in Atonik-treated plants and this increase ranged from 0.5 to as high as55.5%. The greater intensity of photosynthesis corresponded wellwith the significantly lowered stomatal resistance. The effect ofAtonik on chlorophyll content in A. thaliana was not uniform.

Measurements taken seven days after the treatment revealed thebiostimulant’s positive effect on this parameter, but 14 days afterthe Atonik application a greater chlorophyll content was recordedin 50 and 40% of MWC. Atonik also influenced parameters ofchlorophyll a fluorescence, especially 14 days after its application,when the negative effects of drought stress were more evident(Table 7).

The intensity of transpiration increased after Atonik treatment(Table 8). RWC was either only lowered slightly or, at higherdrought levels, even increased due to biostimulant application.Plants sprayed with Atonik uptake more water from the medium(Table 8).

Optimal and Pt stress conditionsTreatment with Atonik, independently from Pt concentration,had a positive effect on A. thaliana plants. The area and number ofleaves were greater than in the control by 8.6–15.1 and 0.2–35.5%,respectively (Table 9). Only plants grown in the Pt-free medium





FIGURE 1 | Effect of Atonik on selected parameters of chlorophyll a

fluorescence (Fv/Fm and PI) in oilseed rape cv. Lisek plants grown underfield conditions during the 2007 (A) and 2008 (B) vegetation seasons.Presented data are mean ± SE, n = 24. *Values differ significantly atα = 0.05 as determined by LSD of t-Student test.

had a decreased number of leaves after Atonik application. Thebiostimulant had a positive effect on biomass accumulation in theaboveground parts of the plants exposed to Pt at concentrationsof 2.5 and 25 μM, and the range of this increase amounted to 13–14.5%. In the case of 50 μM, a positive effect was not recorded.Atonik always increased the fresh weight and dry matter of roots(Table 9).

Intensity of photosynthesis was greater (up to 17.5%) andstomatal resistance was lower (up to 42.5%) in Atonik-treatedplants (Table 10). The biostimulant also had a positive effect onthe chlorophyll content in leaves, which was higher by 5.1–13.0%.Treatment with Atonik raised the values of Fv/Fm and P.I. inplants exposed to Pt ions (Table 10).

Treatment with Atonik always increased the intensity of tran-spiration, which was especially evident in 2.5 μM of Pt (Table 11).Effect of Atonik on RWC was marginal as the biostimulantincreased this parameter by 3–4% in two lower Pt concentrations,and decreased it by 2% in the highest (Table 11).

Membrane injuries were reduced in biostimulant treatedplants (Table 11). After application of Atonik, the level of mem-brane injuries decreased by 9.5–13.8% in roots and 0.5–1.7% inleaves (Table 11).

DISCUSSIONEFFECT OF ATONIK ON GROWTH AND DEVELOPMENTThe results of this study have clearly demonstrated that Atonikaffects all stages of plant development. Changes caused by the

biostimulant application are recorded from seed germinationand seedling growth (our other study on Atriplex hortensis,Lolium perenne, and Sinapis alba, data not shown) through thewhole ontogenesis. The positive effect of Atonik on germina-tion and seedling growth has been reported by Djanaguiramanet al. (2005a) and Kozak et al. (2008b). This can be explainedby the fact that phenolic compounds, which are components ofAtonik, interact with gibberellins, which promote seed germi-nation (Taiz and Zeiger, 2002). Fully developed plants treatedwith a biostimulant are more advanced in growth and devel-opment, which has been shown in this work on A. thalianaand oilseed rape. A. thaliana plants had an increased leaf areaand better-developed root system. The stimulation of elonga-tive growth, as a result of the application of Atonik, might beattributed to the greater concentration and/or activity of aux-ins (Djanaguiraman et al., 2004a, 2005b). Plants treated with abiostimulant are characterized as having a higher inhibition ofIAA oxidase, which ensures greater activity of naturally synthe-sized auxins (Stutte and Clark, 1990) and a greater number ofhigh-affinity binding sites of IAA (Libbenga and Mennes, 1987).Feverfew (Gruszczyk and Berbec, 2004), cotton (Djanaguiramanet al., 2005a, 2009), tomato (Djanaguiraman et al., 2004b, 2005a),maize (Michalski et al., 2008), and soya (Kozak et al., 2008a) areall taller after the Atonik application. The biostimulant stimu-lates the growth of shoots in sweet pepper (Panajotov et al., 1997)and roots in cotton (Djanaguiraman et al., 2005a) and ginseng(Kołodziej, 2004). The promotion of leaf development is notedin cotton and tomato (Djanaguiraman et al., 2005b, 2009) andsweet pepper (Panajotov et al., 1997). Other biostimulants alsostimulate plant growth. For example bio-algeen S90 increases theheight of tomato plants (Dobromilska and Gubarewicz, 2008).The length of shoots has been positively influenced by variousbiostimulants in bell pepper, raspberry, and apple (Basak andMikos-Bielak, 2008; Ochmian et al., 2008; Stepowska, 2008a). BioJodis, Goëmar Goteo, Bio-algeen S90 and Resistim stimulate rootgrowth in tomato (Kossak and Dyki, 2008). A greater number ofleaves and/or their area have been recorded in tomato treated withBio-algeen S90 (Dobromilska and Gubarewicz, 2008), apple withKelpak (Basak and Mikos-Bielak, 2008) and bell pepper with fourdifferent biostimulants (Stepowska, 2008a).

However, in literature there is also data indicating a lack of pos-itive effects of biostimulants on plant growth. Malarz et al. (2008)demonstrate the marginal influence of Atonik on the height ofspring rape. Atonik did not affect the growth of oilseed rape(Budzynski et al., 2008), bell pepper (Csizinszky, 2001), or maize(Ksiezak, 2008) at all.

Atonik-treated plants are more advanced in generative devel-opment. In this study, the biostimulant increased the numberof inflorescences, pods and seeds. This was true for A. thalianaand oilseed rape, irrespective of whether the plants were grownin the field or in growth chambers, no matter if under optimalor stress conditions. These results confirmed previous findingsby Budzynski et al. (2008) and Malarz et al. (2008), who alsodemonstrate the positive effect of Atonik on the generative devel-opment of oilseed rape. Atonik also increases the number ofpods and seeds in soya (Gulluoglu et al., 2006; Kozak et al.,2008a), flowers and bolls in cotton (Djanaguiraman et al., 2005b),






Table 4 | Effect of Atonik on the total and marketable yield of cucumber fruit, content of dry matter, soluble solids, nitrates, phosphorus,

potassium, and calcium.

Cultivar Total yield (kg m−2) Mean for cultivar Marketable yield (kg m−2) Mean for cultivar

Control Atonik Control Atonik

Octopus F1 6.43 ba 7.52 a 6.97 b 3.94 b 4.76 b 4.35 b

Opera F1 7.74 a 7.17 a 7.45 ab 5.71 a 5.20 a 5.45 a

Sonate F1 8.35 a 8.26 a 8.30 a 6.05 a 5.95 a 6.00 a

Mean for treatments 7.51 a 7.65 a 5.23 a 5.30 a

Dry matter (%) Soluble solids (%)

Octopus F1 4.22 a 4.75 a 4.48 a 4.10 a 4.20 a 4.15 a

Opera F1 4.94 a 4.74 a 4.84 a 4.10 a 4.47 a 4.28 a

Sonate F1 4.50 a 5.20 a 4.85 a 4.13 a 4.23 a 4.18 a

Mean for treatments 4.56 a 4.90 a 4.11 b 4.30 a

Nitrates (mg 100 g−1 FW) Phosphorus (mg 100 g−1 FW)

Octopus F1 12.96 c 13.99 b 13.47 a 16.14 b 18.24 a 17.19 a

Opera F1 13.10 b 15.01 a 14.05 a 15.93 b 17.82 b 16.87 ab

Sonate F1 13.79 b 13.23 b 13.51 a 11.81 c 12.29 c 12.05 c

Mean for treatments 13.28 b 14.08 a 14.63 b 16.11 a

Potassium (mg 100 g−1 FW) Calcium (mg 100 g−1 FW)

Octopus F1 219.84 a 215.78 a 217.81 a 6.96 b 5.77 b 6.36 b

Opera F1 209.79 a 209.38 a 209.58 a 6.03 b 6.16 b 6.09 b

Sonate F1 196.35 b 198.88 b 197.61 b 13.67 a 13.67 a 13.67 a

Mean for treatments 208.66 a 208.0 a 8.89 a 8.53 a

Presented data are mean, n = 3.aData in columns followed by the same letter do not differ significantly as based on the HSD of the Tukey test at confidence level of 95%.

flowers and fruits in tomato (Djanaguiraman et al., 2004a), andinflorescences in feverfew (Gruszczyk and Berbec, 2004). Abovecorresponds well with works of Górnik and Grzesik (2002, 2005),who found that Atonik improves the generative development ofChina aster, but only when applied during flowering. A greaternumber of flowers and fruits has also been reported in tomatoand apple plants treated with Bio-algeen S-90 and Frigocur,respectively (Basak and Mikos-Bielak, 2008; Dobromilska andGubarewicz, 2008). Goëmar BM 86 stimulates fruit growth inpears (Błaszczyk, 2008) and ripening in raspberries (Krok andWieniarska, 2008).

In contrast, Krawiec (2008) found an ambiguous effect ofsimultaneous treatment with Goëmar BM 86 and Atonik on thenumber of fruits in chokeberries. Atonik did not affect the sizeand diameter of strawberry fruits (Miranda-Stalder et al., 1990)or the number of grains in the cob and size of the cob in maize(Ksiezak, 2008).

EFFECT OF ATONIK ON BIOMASS ACCUMULATION AND YIELDINGThis study’s results have shown, that the faster growth and devel-opment of Atonik-treated plants is associated with a greaterbiomass accumulation. After the application of the biostimulant,the fresh weight and dry matter of whole A. thaliana and oilseedrape plants, as well as their particular organs, were greater. In the

case of oilseed rape, this effect was more pronounced in the 2007vegetative season in which plants experienced a spring frost. It isworth mentioning that the increase of biomass accumulated ingenerative organs was greater than in vegetative ones, which alsosupported the hypothesis mentioned above concerning the pro-motion of generative development. A greater biomass accumula-tion in oilseed rape sprayed with Atonik has also been recordedby Becka et al. (2004). Similar results are recorded for cottonand tomato (Djanaguiraman et al., 2004b, 2005a), goldenrod(Kołodziej, 2008), Amaranth sp. (Wrochna et al., 2008), and com-mon osier (Harasimowicz-Hermann and Czyz, 2008). A stimu-lation of dry-matter accumulation in the roots and abovegroundorgans of oilseed rape treated with Route has been reported byKrawczyk and Skoczynski (2008). Bio-algeen S-90 increases thedry matter of tomato fruits (Dobromilska and Gubarewicz, 2008)and Goëmar Goteo positively affects biomass accumulation in let-tuce (Kowalczyk and Zielony, 2008) and nappa cabbage (Gajewskiet al., 2008). Stepowska (2008a) has recorded a greater weightof whole plants and separately of roots and leaves in bell pep-per treated with different biostimulants. The increase in biomassaccumulation resulting from biostimulant treatment is not usu-ally very spectacular and ranges from just a little to 20%, butmuch higher values are also reported, as in the case of feverfewand ginseng plants in which the application of Atonik results in





FIGURE 2 | Effect of Atonik on growth and development of A. thaliana

plants grown under drought stress conditions (40% MWC).

the increase in biomass of 54 and 51.5% (fresh weight and drymatter) and 43 and 61% (fresh weight of roots and abovegroundorgans) respectively (Gruszczyk and Berbec, 2004; Kołodziej,2004).

The increased biomass accumulation after Atonik applicationusually resulted in a higher yield. In this study the biostimu-lant increased the yield of oilseed rape, but only when it wasapplied as a single spray. This has also been shown in oilseedrape by Budzynski et al. (2008) and Malarz et al. (2008), aswell as in many other species, such as beetroot (Cerný et al.,2002; Kositorna and Smolinski, 2008), potato (Czeczko andMikos-Bielak, 2004; Sawicka and Mikos-Bielak, 2008), cotton(Djanaguiraman et al., 2005b, 2009; Bynum et al., 2007), maize(Michalski et al., 2008), soya (Kozak et al., 2008a), tomato(Djanaguiraman et al., 2004a,b; Gajc-Wolska et al., 2010), apple(Basak and Mikos-Bielak, 2008), common chicory (Cerný et al.,2008), leek and celery (Czeczko and Mikos-Bielak, 2004), andraspberries (Grajkowski and Ochmian, 2007). Other biostim-ulants also increase yield and this has been reported for agreat number of crops, such as apple, bell pepper, cereals, let-tuce, lupine, maize, mustard, nappa cabbage, pea, potato, rasp-berry, and strawberry (Abetz and Young, 1983; Dobromilska andGubarewicz, 2008; Gajewski et al., 2008; Kossak and Dyki, 2008;Kowalczyk and Zielony, 2008; Matysiak and Kaczmarek, 2008;Ochmian et al., 2008; Sas-Paszt et al., 2008; Stepowska, 2008b;Wrona and Misiura, 2008; Khan et al., 2009).

However, there are reported studies showing that biostim-ulants have either a minor, no influence on yield or even anegative effect. The lack of a positive effect of Atonik on yieldhas been recorded here in cucumber and earlier reported byMiranda-Stalder et al. (1990), Csizinszky (2001), Krawiec (2008),

and Ksiezak (2008) in strawberries, bell pepper, chokeberriesand maize. Basak and Mikos-Bielak (2008) showed that Frigocur,Kelpak, and Help even negatively affect the yield of apples.

The effect of biostimulants on biomass accumulation and yieldmay depend on a number of environmental factors. In litera-ture the emphasis is on the influence of the cultivar, preparationconcentration and term of its application, growing conditions,fertilization employed, and location (Basak and Mikos-Bielak,2008; Łyszkowska et al., 2008; Maciejewski et al., 2008; Sas-Pasztet al., 2008; Gajc-Wolska et al., 2009, 2012).

EFFECT OF ATONIK ON PHOTOSYNTHETIC APPARATUSStimulated biomass production and yield recorded for manyspecies are attributed to a more efficient photosynthetic appara-tus in plants sprayed with Atonik. This has been shown in thisstudy for A. thaliana and oilseed rape. Plants treated with Atonikhad higher (i) leaf area, (ii), chlorophyll content, (iii) intensityof photosynthesis, and (iv) values of chlorophyll a fluorescenceparameters. In our preliminary studies on wheat also increase inLAI (Leaf Area Index, data not shown) was recorded.

In this work, the biostimulant increased the chlorophyll con-tent in both Brassicaceae species examined and under all exper-imental conditions. It is worth mentioning that this increase inthe case of oilseed rape was more evident at the end of thegrowing season, which may suggest that Atonik either promotesde novo chlorophyll biosynthesis or slows down its degradation,delaying the aging processes. A similar result was reported byDjanaguiraman et al. (2009) in cotton. A greater chlorophyll con-tent in plants treated with Atonik was recorded also in commonosier (Wróbel and Wozniak, 2008), Amaranthus sp. (Wrochnaet al., 2008) and cotton (Djanaguiraman et al., 2009). Four dif-ferent biostimulants increased the content of chlorophyll in bellpepper (Stepowska, 2008a). In contrast to the above, Kowalczyket al. (2008) did not find that Atonik and Aminoplant hada positive effect on the content of chlorophyll in lettuce, andKrajewska and Latkowska (2008) even demonstrated a reduc-tion of chlorophyll content in hosta and bergenia treated withSiapton.

In A. thaliana and oilseed rape plants treated with Atonik,the intensity of photosynthesis was greater, which is in the linewith the results of Borowski and Blamowski (2009), Wróbel andWozniak (2008) and Djanaguiraman et al. (2009) on basil, com-mon osier and cotton. A new discovery from this study hasbeen that the positive effect of Atonik on the intensity of pho-tosynthesis may last up to 7 weeks, which is much longer thanpreviously believed. According to the manufacturer of Atonik,its working time was estimated to be a maximum of 2–3 weeks.A higher intensity of photosynthesis could be explained, at leastpartially, by lowered stomatal resistance (or increased stomatalconductance), which ensures an easier and greater CO2 flow tochloroplast. Increased stomatal conductance has been reportedfor basil plants treated with Atonik (Borowski and Blamowski,2009) and cotton with PGR-IV (Zhao and Oosterhuis, 1997).Atonik accelerates the transport of photoassimilates within cellsand between them to various tissues and organs (Yamaki et al.,1953). Wilson and Kaczmarek (1993) show that the phospho-rylated form of sodium para-nitrophenolate reduces the activity






Table 5 | Effect of Atonik on selected morphological parameters of A. thaliana plants grown under optimal and drought stress conditions.

MWC (%) Height (cm plant−1) Number of (inflorescence plant−1) Number of (pod plant−1) Leaf area (cm2 plant−1)

−Atonik +Atonik −Atonik +Atonik −Atonik +Atonik −Atonik +Atonik

60 34.95 (± 2.42) 42.92 (± 1.17) 27.50 (± 3.18) 43.00 (± 5.86) 12.00 (± 2.09) 41.50 (± 9.83) 164.16 (± 9.53) 191.51 (± 4.60)

50 24.67 (± 1.91) 29.17 (± 1.60) 20.50 (± 2.93) 23.50 (± 1.53) 7.75 (± 2.00) 15.00 (± 6.84) 130.22 (± 10.32) 134.25 (± 4.81)

40 20.95 (± 0.61) 26.12 (± 2.07) 18.50 (± 2.73) 26.25 (± 4.13) 0.50 (± 0.25) 2.75 (± 0.69) 104.06 (± 2.15) 110.03 (± 5.23)

30 14.12 (± 0.71) 21.47 (± 1.49) 18.50 (± 1.49) 25.75 (± 2.68) 1.75 (± 0.88) 1.00 (± 0.29) 77.02 (± 4.20) 110.33* (± 3.26)

20 11.72 (± 0.82) 13.37 (± 1.29) 15.25 (± 1.66) 15.00 (± 1.24) 0.50 (± 0.25) 1.50 (± 0.75) 81.02 (± 4.07) 88.57 (± 4.46)

Data are Mean ± SE, n = 5.*Values differ significantly at α = 0.05 as determined by LSD of t-Student test.

Table 6 | Effect of Atonik on the fresh matter of the whole aboveground part, inflorescence and rosette of A. thaliana plants grown under

optimal and drought stress conditions.

MWC (%) Aboveground part Inflorescence with pods Rosette

−Atonik +Atonik −Atonik +Atonik −Atonik +Atonik

FRESH WEIGHT g PLANT−1

60 18.28 (± 0.97) 23.97 (± 0.62) 5.49 (± 0.57) 8.28 (± 0.79) 12.78 (± 0.49) 15.69 (± 0.48)

50 16.07 (± 0.93) 16.48 (± 0.89) 4.96 (± 0.29) 4.74 (± 0.33) 11.12 (± 0.94) 11.74 (± 0.89)

40 13.14 (± 0.56) 16.06 (± 0.31) 3.79 (± 0.38) 4.36 (± 0.33) 9.35 (± 0.20) 11.70 (± 0.38)

30 9.70 (± 0.20) 14.15* (± 0.20) 2.36 (± 0.15) 4.11 (± 0.41) 7.34 (± 0.08) 10.04* (± 0.21)

20 9.32 (± 0.40) 10.36 (± 0.47) 1.34 (± 0.16) 2.12 (± 0.23) 7.97 (± 0.24) 8.50 (± 0.24)

DRY MATTER g PLANT−1

60 2.03 (± 0.10) 2.58 (± 0.05) 0.68 (± 0.07) 1.07 (± 0.09) 1.35 (± 0.05) 1.51 (± 0.05)

50 1.94 (± 0.11) 1.96 (± 0.10) 0.61 (± 0.05) 0.61 (± 0.04) 1.33 (± 0.09) 1.35 (± 0.09)

40 1.68 (± 0.08) 1.99 (± 0.06) 0.48 (± 0.05) 0.59 (± 0.05) 1.20 (± 0.03) 1.41 (± 0.05)

30 1.19 (± 0.06) 1.76 (± 0.01) 0.30 (± 0.02) 0.56 (± 0.04) 0.97 (± 0.00) 1.21 (± 0.05)

20 1.25 (± 0.02) 1.35 (± 0.05) 0.17 (± 0.02) 0.30 (± 0.03) 1.07 (± 0.00) 1.05 (± 0.04)

Data are Mean ± SE, n = 5.*Values differ significantly at α = 0.05 as determined by LSD of t-Student test.

of cation channels (Ca2+, K+, and Na+) by inhibiting the activ-ity of the enzyme tyrosine phosphatase. A decreased activity inthe cation channel causes the reduction of Ca2+ concentrationin the cells, which results in the increase of cytoplasm move-ment (Roberts and Harmon, 1992). The above is in line withOosterhuis and Robertson (2000), who suggest that the increasedphotosynthesis in cotton treated with PGR-IV is related to aquicker transport of assimilates from its source (leaves) to varioussinks.

Atonik also has a positive effect on the parameters of chloro-phyll a fluorescence. The values of Fv/Fm and P.I were usuallyhigher in Atonik-treated A. thaliana plants. The positive effectof this biostimulant on chlorophyll a fluorescence has previ-ously been reported by Djanaguiraman et al. (2009) in cotton.In contrast to the above, Gawlik and Gołebiowska (2008) recorddecreased values of Fv/Fm in pea plants sprayed with humicacids.

It should be pointed out that although the level of bene-ficial influence on particular/parameters of the photosyntheticapparatus is not very spectacular, it has to be taken into con-sideration that they “work additively.” Photosynthesis takes placeover several hours a day during most of the sunny days of the

vegetation season, which, together with the positive effects onother processes, substantially contributes to greater final plantproductivity.

EFFECT OF ATONIK ON PLANT WATER STATUSApplication of Atonik also affects a plant’s water status. Thelowered stomatal resistance earlier discussed leads to higherintensity of transpiration in A. thaliana and oilseed rape plants,as reported by Wróbel and Wozniak (2008), Borowski andBlamowski (2009), and Zhao and Oosterhuis (1997). Increasedtranspiration intensity means greater water loss by plants and,as a consequence, it can be expected that RWC should belower, especially in A. thaliana plants grown under droughtstress conditions. Contrary to this expectation, RWC was almostunchanged or, in some cases, even slightly higher. This resultcan be explained by the improved water uptake after Atonikapplication, as shown here by daily pot weighing, which isrelated to a better-developed root system, both in terms oflength and biomass. Improved RWC in biostimulant-sprayedplants has also been reported by Wrochna et al. (2008) inAmaranthus sp. and Wróbel and Wozniak (2008) in commonosier.





Tab

le7

|E

ffect

of

Ato

nik

on

inte

nsit

yo

fp

ho

tosyn

thesis

,sto

mata

lre

sis

tan

ce,

ch

loro

ph

yll

co

nte

nt

an

dsele

cte

dp

ara

mete

rso

fch

loro

ph

yll

afl

uo

rescen

ce

of

A.th

ali

an

aL.p

lan

tsg

row

n

un

der

op

tim

alan

dd

rou

gh

tstr

ess

co

nd

itio

ns.

MW

CTerm

Ph

oto

syn

thesis

(µm

ol

CO

2m

−2s−1

)S

tom

ata

lre

sis

tan

ce

(scm

−1)

Ch

loro

ph

yll

co

nte

nt

(rela

tive

valu

es)

Fv/F

mP.

I.

(%)

(Days)

−Ato

nik

+Ato

nik

−Ato

nik

+Ato

nik

−Ato

nik

+Ato

nik

−Ato

nik

+Ato

nik

−Ato

nik

+Ato

nik

607

7.34

(±0.

17)

8.97

*(±

0.12

)4.

85(±

0.51

)0.

86*

(±0.

02)

11.2

5(±

0.31

)11

.61

(±0.

41)

0.83

2(±

0.00

1)0.

833

(±0.

002)

1.77

(±0.

06)

1.67

(±0.

06)

147.

57(±

0.19

)10

.38*

(±0.

05)

1.83

(±0.

04)

0.44

*(±

0.01

)11

.31

(±0.

85)

11.0

8(±

0.98

)0.

828

(±0.

001)

0.82

8(±

0.00

1)2.

79(±

0.06

)2.

69(±

0.07

)

507

6.77

(±0.

24)

10.5

4*(±

0.22

)6.

07(±

0.52

)0.

72*

(±0.

02)

10.7

9(±

0.55

)11

.36

(±0.

55)

0.82

9(±

0.00

1)0.

838*

(±0.

001)

1.49

(±0.

07)

1.82

*(±

0.02

)

148.

07(±

0.23

)8.

74(±

0.15

)2.

34(±

0.17

)1.

01*(

±0.

04)

10.9

1(±

1.19

)12

.37

(±1.

20)

0.82

1(±

0.00

2)0.

832

(±0.

001)

2.39

(±0.

15)

2.79

(±0.

09)

407

9.02

(±0.

23)

9.94

(±0.

34)

2.97

(±0.

35)

0.72

*(±

0.01

)12

.50

(±0.

32)

12.9

8(±

0.35

)0.

823

(±0.

003)

0.83

3(±

0.00

1)1.

48(±

0.07

)1.

56(±

0.05

)

147.

71(±

0.37

)8.

45(±

0.43

)3.

00(±

0.31

)1.

10*

(±0.

13)

10.3

3(±

0.75

)10

.71

(±1.

26)

0.82

0(±

0.00

2)0.

829

(±0.

002)

2.55

(±0.

17)

2.64

(±0.

14)

307

7.19

(±0.

12)

7.21

(±0.

09)

2.52

(±0.

16)

1.02

*(±

0.04

)11

.45

(±0.

39)

10.9

2(±

0.30

)0.

827

(±0.

002)

0.81

5*(±

0.00

2)1.

56*(

±0.

06)

1.08

*(±

0.06

)

148.

87(±

0.31

)7.

40(±

0.20

)1.

47(±

0.11

)0.

97*(

±0.

05)

12.5

4(±

1.44

)10

.03

(±1.

60)

0.81

8(±

0.00

3)0.

825

(±0.

003)

2.66

(±0.

21)

2.26

(±0.

14)

207

7.04

(±0.

24)

7.98

(±0.

44)

3.05

(±0.

29)

1.31

*(±

0.07

)12

.70

(±0.

34)

14.7

1(±

0.37

)0.

821

(±0.

002)

0.81

4(±

0.00

3)1.

40(±

0.05

)1.

13(±

0.08

)

144.

96(±

0.40

)7.

10(±

0.38

)2.

13(±

0.03

)1.

04*

(±0.

04)

14.6

5(±

2.35

)13

.82

(±1.

27)

0.81

4(±

0.00

3)0.

819

(±0.

002)

2.22

(±0.

13)

2.46

(±0.

16)

Pres

ente

dda

taar

eM

ean

±S

E,n

=15

(gas

exch

ange

and

chlo

roph

yllc

onte

nt)o

r10

(chl

orop

hyll

aflu

ores

cenc

e).

* Val

ues

diffe

rsi

gnifi

cant

lyat

α=

0.05

asde

term

ined

byLS

Dof

t-S

tude

ntte

st.

It is worth noticing that simultaneously with more efficientwater uptake from soil, plants are also taking up more nutrients,as demonstrated by Stutte et al. (1987) and Oosterhuis (2008).

EFFECT OF ATONIK ON PLANT QUALITYAtonik changes the chemical composition of cucumber fruits,positively in the case of soluble solids and phosphorus, but neg-atively in terms of nitrates and calcium. In literature there isdata that the application of Atonik increases the content of car-bohydrates (Czeczko and Mikos-Bielak, 2004; Djanaguiramanet al., 2005a; Kositorna and Smolinski, 2008), crude fat (Malarzet al., 2008), amino acids (Djanaguiraman et al., 2005a) pro-teins (Czeczko and Mikos-Bielak, 2004; Djanaguiraman et al.,2005a; Oosterhuis, 2008), but decreases the level of nitrates(Kowalczyk et al., 2008). On the other hand, Atonik may decreasethe concentration of vitamin C (Czeczko and Mikos-Bielak, 2004;Grajkowski and Ochmian, 2007).

EFFECT OF ATONIK ON THE MITIGATION OF STRESS EFFECTSThere is common opinion that Atonik mitigates effect of stressconditions. This study proved that the application of Atonikdiminished the negative impact of drought and noble metalstresses in A. thaliana and enhanced the recovery from the latespring frost in oilseed rape. A. thaliana plants grown with awater deficit and Pt stresses and treated with a biostimulanthad accelerated growth and development, accumulated morebiomass and all studied physiological processes were stimulated.Protective effect of Atonik was especially evident in the case ofphotosynthetic apparatus. For example in oilseed rape grownin the 2007 season, when the late spring frost occurred, Atonikimproved chlorophyll a fluorescence parameters. Chlorophyll afluorescence is informative tool to analyze and understand plant’sresponse to fluctuations in environmental conditions. Higherintensity of photosynthesis was recorded in A. thaliana plantsgrown under drought conditions. One of the first responses ofplants to drought stress is the closing of stomata, a process con-trolled by, among other, ABA (Blatt, 2000; Schroeder et al., 2001;Shinozaki and Yamaguchi-Schinozaki, 2007). A decreased levelof free ABA after application of Atonik has been shown in otherstudies conducted by the authors in A. thaliana plants grown withwater deficit (Przybysz et al., 2010). Changes in ABA regulation bylowering its concentration resulted in more efficient gas exchangeand stimulated growth in stress conditions recorded in this work.

More evidence that Atonik protects plants against the nega-tive effects of stress was shown in this work in the decreased levelof plasma membrane injuries caused by Pt, both in the rootsand leaves of A. thaliana. Similar results were obtained in previ-ous work on plants exposed to Cd2+ (Gawronska et al., 2008).The reduction of membrane injuries in the case of both met-als was more pronounced in roots, which were in direct contactwith toxic elements. A decrease in plasma membrane injurieshas also been found in Atonik-treated Amaranthus sp. (Wrochnaet al., 2008), basil (Borowski and Blamowski, 2009), and cotton(Djanaguiraman et al., 2009).

The protective role of Atonik has also been recorded inthe case of heavy metals in the example of Cd2+ (Gawronskaet al., 2008), salinity (Wrochna et al., 2008), spring frost






Table 8 | Effect of Atonik on intensity of transpiration, RWC, and water uptake of A. thaliana plants grown under optimal and drought stress

conditions.

MWC (%) Term (Days) Transpiration (µmol CO2 m−2 s−1) RWC (%) Water uptake (ml pot−1)

−Atonik +Atonik −Atonik +Atonik −Atonik +Atonik

60 7 2.36 (± 012) 5.55* (± 0.07)

14 3.66 (± 0.02) 8.21* (± 0.10) 90.98 (± 0.74) 87.55 (± 1.28) 26.2 (± 0.56) 29.0 (± 0.50)

50 7 1.98 (± 0.10) 6.66* (± 0.20)

14 3.63 (± 0.21) 5.40* (± 0.10) 89.11 (± 1.40) 87.77 (± 0.84) 21.5 (± 0.53) 23.0 (± 0.60)

40 7 3.62 (± 0.22) 5.94* (± 0.04)

14 3.37 (± 0.19) 5.88* (± 0.32) 92.18 (± 0.66) 90.30 (± 0.31) 21.3 (± 0.45) 22.5 (± 0.41)

30 7 3.50 (± 0.13) 4.99* (± 0.05)

14 4.70 (± 0.19) 5.71* (± 0.10) 82.28 (± 0.93) 92.91∗ (± 0.41) 16.1 (± 0.44) 19.2* (± 0.43)

20 7 3.41 (± 0.17) 4.94* (± 0.15)

14 3.78 (± 0.04) 4.78 (± 0.42) 83.22 (± 3.10) 85.55 (± 2.11) 15.2 (± 0.39) 16.3 (± 0.26)

Presented data are Mean ± SE, n = 15 (intensity of transpiration) or 5.*Values differ significantly at α = 0.05 as determined by LSD of t-Student test.

(Basak and Mikos-Bielak, 2008), and heat (Gulluoglu et al.,2006). Górnik et al. (2007) and Górnik and Grzesik (2008)recorded an increased tolerance of grape cuttings to extremetemperatures and water deficit after treatment with a few biostim-ulants. Since many defense mechanisms against different unfa-vorable conditions, especially of abiotic origin, are very much thesame, it can be assumed that Atonik probably also decreases thenegative effects of other stresses not mentioned in this work.

Most of the stresses may induce the appearance of excessiveamounts of reactive oxygen species (ROS) and consequently theexacerbation of oxidative stress (Iturbe-Ormaetxe et al., 1998).Discussed above reduction of membrane injuries may reducecreating of ROS in plants. Moreover, it has been reported thatthe application of Atonik contributes to a decreased level ofoxidative stress by increasing (i) the activity of anti-oxidizing sys-tem enzymes: ascorbate peroxidase, catalase, glutathione reduc-tase, and (ii) total antioxidative capacity to a greater extentthan the increase in anion-radical level (Wrochna et al., 2008;Djanaguiraman et al., 2004a, 2005a,b, 2009). Atonik also posi-tively affects the production of proline and polyols, two impor-tant compatible metabolites involved in anti-stress mechanisms(Djanaguiraman et al., 2004b, 2009).

All changes presented above have probably their origin inmodified, after Atonik treatment, profile of gene expression. Inthe literature some studies report that after the application ofbiostimulants expression of genes related to defense mechanism isupregulated. The treatment of A. thaliana plants exposed to freez-ing stresses with algae extract result in changes of expression inabout 5% (1113) of all A. thaliana genes (Nair et al., 2012). About2% (463 genes) of the differentially expressed genes are upregu-lated and 3% (650 genes) downregulated. The authors report thatsome of these genes were involved in the plant’s defense mecha-nisms (Nair et al., 2012). The application of algal extracts priorto pathogen infection in alfalfa cause upregulation of 152 genes,mostly plant defense genes, such as those involved in phytoalexin,PR proteins, cell wall proteins, and oxylipin pathways (Cluzet

et al., 2004). In A. thaliana grown under salt stress and treatedwith Aminoplant, Cambri et al. (2008) demonstrate changes inexpression of a few genes responsible for the plant’s defensemechanisms.

There is a commonly held view, as also demonstrated in thiswork, that the positive impact of biostimulants is more evi-dent and that the potential of these compounds can be fullyexploited only when plants are grown under stressful conditions,while under optimal conditions their positive effect is sometimesmarginal (Budzynski et al., 2008; Krawiec, 2008; Maciejewskiet al., 2008) or even not reported at all (Csizinszky, 2001). Possibleprotective effect of biostimulants depends also on many other, notdiscussed here factors, mostly the level and duration of stressesand moment of Atonik application.

CONCLUSIONSThe biostimulant Atonik affects every level of a plant’s biologicalorganization in terms of structure and function, from canopy andwhole plant, via particular organs and cells, to physiological andbiochemical processes.

(1) Atonik stimulates plant growth and development, particu-larly generative.

(2) Biomass accumulation, both fresh weight and dry matter, andyield production are stimulated by Atonik due to a higherefficiency of the photosynthetic apparatus manifested by (i)a higher leaf area, (ii) a higher chlorophyll content, (iii)greater intensity of photosynthesis, and (iv) an improvementof chlorophyll a fluorescence parameters.

(3) Despite higher transpiration and lower stomatal resistance,RWC was unchanged in Atonik-treated plants due to the pro-motion of root development and consequently an increasedwater uptake.

(4) The effect of Atonik on the quality and chemical composi-tion of fruits was diverse and depended on the parametermeasured and cultivar examined.





Tab

le9

|E

ffect

of

Ato

nik

on

the

nu

mb

er

an

dare

ao

fle

aves,

an

db

iom

ass

accu

mu

lati

on

inA

.th

alia

na

pla

nts

exp

osed

toP

tio

ns.

Co

mb

inati

on

Nu

mb

er

of

(leaves

pla

nt−

1)

Leaves

are

a(c

m2

pla

nt−

1)

Fre

sh

weig

ht

(gp

lan

t−1)

Dry

matt

er

(mg

pla

nt−

1)

−Ato

nik

+Ato

nik

−Ato

nik

+Ato

nik

Ab

oveg

rou

nd

part

sR

oo

tsA

bo

veg

rou

nd

part

sR

oo

ts

−Ato

nik

+Ato

nik

−Ato

nik

+Ato

nik

−Ato

nik

+Ato

nik

−Ato

nik

+Ato

nik

014

3.00

(±1.

61)

128.

00(±

2.92

)22

2.78

(±0.

74)

271.

26(±

2.17

)8.

58(±

0.50

)9.

61(±

0.17

)2.

59(±

0.12

)2.

72(±

0.02

)88

0(±

50)

990

(±40

)14

5(±

12.5

)14

5(±

1)

2.5

μM

Pt

145.

00(±

3.22

)196

.50∗

(±3.

93)

274.

28(±

7.83

)30

0.05

(±3.

59)

8.88

(±0.

37)

10.0

7(±

0.66

)3.

82(±

0.07

)4.

63(±

0.55

)91

0(±

37)

1030

(±65

)19

5(±

2.5)

205

(±12

.5)

25μ

MP

t16

1.50

(±8.

58)

202.

50(±

1.94

)21

7.5

(±3.

04)

236.

22(±

3.82

)7.

05(±

0.47

)8.

07(±

0.34

)2.

50(±

0.03

)2.

89(±

0.18

)83

0(±

47)

950

(±34

)14

0(±

2)15

0(±

5)

50μ

MP

t19

3.00

(±2.

29)

193.

50(±

2.51

)21

9.11

(±0 .

34)

252.

20(±

9.44

)7.

12(±

0.64

)6.

97(±

0.50

)2.

44(±

0.22

)2.

95(±

0.34

)85

0(±

64)

840

(±50

)11

0(±

5)14

0(±

15)

Pres

ente

dda

taar

eM

ean

±S

E,n

=5.

* Val

ues

diffe

rsi

gnifi

cant

lyat

α=

0.05

asde

term

ined

byLS

Dof

t-S

tude

ntte

st.

Tab

le10

|E

ffect

of

Ato

nik

on

inte

nsit

yo

fp

ho

tosyn

thesis

,sto

ma

tal

resis

tan

ce

,ch

loro

ph

yll

co

nte

nt

an

dse

lecte

dp

ara

me

ters

of

ch

loro

ph

yll

afl

uo

rescen

ce

(Fv/F

man

dP.

I.)

of

A.th

ali

an

a

pla

nts

exp

osed

toP

tio

ns.

Co

mb

inati

on

Ph

oto

syn

thesis

(µm

ol

CO

2m

−2s−1

)S

tom

ata

lre

sis

tan

ce

(scm

−1)

Ch

loro

ph

yll

co

nte

nt

(rela

tive

valu

es)

Fv/F

mP.

I.

−Ato

nik

+Ato

nik

−Ato

nik

+Ato

nik

−Ato

nik

+Ato

nik

−Ato

nik

+Ato

nik

−Ato

nik

+Ato

nik

013

.06

(±0.

31)

15.0

3(±

0.12

)1.

80(±

0.07

)0.

99(±

0.01

)13

.18

(±0.

63)

13.5

7(±

0.41

)0.

823

(±0.

002)

0.82

4(±

0.00

1)2.

30(±

0.07

)2.

32(±

0.06

)

2.5

μM

Pt

14.4

9(±

0.22

)16

.37

(±0.

48)

1.56

(±0.

09)

0.90

*(±

0.03

)15

.52

(±0.

35)

17.5

4(±

0.52

)0.

823

(±0.

002)

0.82

6(±

0.00

2)2.

37(±

0.07

)2.

56(±

0.07

)

25μ

MP

t13

.66

(±0.

35)

14.6

6(±

0.34

)1.

56(±

0.04

)1.

34(±

0.04

)13

.07

(±0.

44)

13.4

7(±

0.28

)0.

806

(±0.

002)

0.81

7(±

0.00

1)2.

09(±

0.09

)2.

17(±

0.08

)

50μ

MP

t12

.05

(±0.

34)

14.1

6 *(±

0.33

)2.

00(±

0.16

)1.

95(±

0.11

)12

.46

(±0.

55)

13.1

0(±

0.65

)0.

793

(±0.

004)

0.80

4(±

0.00

2)1.

75(±

0.11

)1.

92(±

0.10

)

Pres

ente

dda

taar

eM

ean

±S

E,n

=15

or10

(chl

orop

hyll

aflu

ores

cenc

e).

* Val

ues

diffe

rsi

gnifi

cant

lyat

α=

0.05

asde

term

ined

byLS

Dof

t-S

tude

ntte

st.






Table 11 | Effect of Atonik on the intensity of transpiration, RWC, and membrane injuries of A. thaliana plants exposed to Pt ions.

Combination Transpiration (µmol CO2 m−2 s−1) RWC (%) Membrane injuries (% of control)

−Atonik +Atonik −Atonik +Atonik Roots Leaves

−Atonik +Atonik −Atonik +Atonik

0 4.75 (± 0.22) 5.98 (± 0.10) 85.57 (± 0.77) 82.15 (± 1.28) 0.00 7.58 0.00 2.74

2.5 μM Pt 4.79 (± 0.26) 7.10* (± 0.14) 85.75 (± 1.12) 89.32 (± 0.58) 21.40 7.64 4.41 3.86

25 μM Pt 4.95 (± 0.09) 5.35 (± 0.10) 88.47 (± 0.69) 90.69 (± 0.76) 22.33 12.81 4.95 3.70

50 μM Pt 4.32 (± 0.19) 4.41 (± 0.17) 90.27 (± 1.29) 88.81 (± 0.60) 31.59 19.21 8.50 6.83

Presented data are Mean ± SE, n = 15 (intensity of transpiration) or 5 (RWC and membrane injuries).*Values differ significantly at α = 0.05 as determined by LSD of t-Student test.

(5) The application of Atonik played simulative role under opti-mal conditions and protective against spring frost, drought,and noble metal stresses.

(6) The positive effect of Atonik is much more pronounced whenplants are growing under stress conditions.

ACKNOWLEDGMENTSThis study has been supported by Arysta LifeScience Poland Ltd.,Asahi Chemical Mfg. Co. Ltd., Japan and Warsaw Plant HealthInitiative FP7-REGPOT-2011-1-286093.

The authors would like to thank the anonymous Reviewers andEditors for their time and effort in improving the quality of thepaper.

Arkadiusz Przybysz conducted the experiments, collected, andanalyzed data on oilseed rape and Arabidopsis and wrote thefirst version of the manuscript. Helena Gawronska designed theexperiments, analyzed the data on oilseed rape and Arabidopsisand corrected the manuscript. Janina Gajc-Wolska conducted theexperiments, collected, and analyzed the data on cucumber andcorrected the manuscript.

REFERENCESAbetz, P., and Young, C. L. (1983). The effect of seaweed extract sprays derived from

Ascophyllum nodosum on lettuce and cauliflower crops. Bot. Mar. 26, 487–492.doi: 10.1515/botm.1983.26.10.487

Arnon, D. I., and Hoagland, D. R. (1940). Crop production in artificial culturesolutions and in soils with special reference to factors influencing yields andabsorption of inorganic nutrients. Soil Sci. 50, 463–471.

Basak, A., and Mikos-Bielak, M. (2008). “The use of some biostimulators on appleand pear trees,” in Monographs Series: Biostimulators in Modern Agriculture:Fruit Crops, ed A. Sadowski (Warsaw: Editorial House Wies Jutra), 7–17.

Becka, D., Vašák, J., Kroutil, P., and Stranc, P. (2004). Autumn growth and develop-ment of different winter oilseed rape variety types at three inputs levels. PlantSoil Environ. 50, 168–174.

Błaszczyk, J. (2008). “Quality of ‘conference’ pears trees as affected by Goëmar BM86 and fruton,” in Monographs Series: Biostimulators in Modern Agriculture: FruitCrops, ed A. Sadowski (Warsaw: Editorial House Wies Jutra), 18–24.

Blatt, M. R. (2000). Cellular signaling and volume control in stomatalmovements in plants. Annu. Rev. Cell Dev. Biol. 16, 221–241. doi:10.1146/annurev.cellbio.16.1.221

Borowski, E., and Blamowski, Z. K. (2009). The effects of triacontanol ‘TRIA’and Asahi SL on the development and metabolic activity of sweet basil(Ocimum basilicum L.) plants treated with chilling. Folia Hort 21, 39–48. doi:10.2478/fhort-2013-0124

Bray, E. A., Bailey-Serres, J., and Weretilnyk, E. (2000). “Response to abioticstresses,” in Biochemistry and Molecular Biology of Plants, eds W. Gruissem,B. Buchanan and R. Jones (Rockville, MD: American Society of PlantPhysiologists), 1158–1249.

Budzynski, W., Dubis, B., and Jankowski, A. (2008). “Response of winter oilseedrape to the biostymulator Asahi SL applied in spring,” in Monographs Series:Biostimulators in Modern Agriculture: Field Crop, ed Z. T. Dabrowski (Warsaw:Editorial House Wies Jutra), 47–55.

Bynum, J. B., Cothren, J. T., Lemon, R. G., Fromme, D. D., and Boman, R. K. (2007).Field evaluation of nitrophenolate plant growth regulator (Chaperone) for theeffect on cotton lint yield. J. Cotton Sci. 11, 20–25.

Calvo, P., Nelson, L., and Kloepper, J. W. (2014). Agricultural uses of plantbiostimulants. Plant soil 383, 3–41. doi: 10.1007/s11104-014-2131-8

Cambri, D., Filippino, L., Apone, F., Arciello, S., Colucci, G., and Portoso, D. (2008).“Effect of Amonoplant® on expression of selected genes in Arabidopsis thalianaL. plants,” in Monographs Series: Biostimulators in Modern Agriculture: GeneralAspects, ed H. Gawronska (Warsaw: Editorial House Wies Jutra), 77–82.

Cerný, I., Pacuta, V., Feckova, J., and Golian, J. (2002). Effect of year and Atonikapplication on the selected sugar beet production and quality parameters.J. Central Eur. Agric. 3, 15–21.

Cerný, I., Pacuta, V., and Kovar, M. (2008). Yield and quality of chicory (Cichoriumintybus L.) in dependence on variety and foliar application of Atonik andPolybor 150. J. Central Eur. Agric. 9, 425–430.

Cluzet, S., Torregrosa, C., Jacquet, C., Lafitte, C., Fournier, J., Mercier, L., et al.(2004). Gene expression profiling and protection of Medicago truncatula againsta fungal infection in response to an elicitor from the green alga Ulva spp. PlantCell Environ. 27, 917–928. doi: 10.1111/j.1365-3040.2004.01197.x

Csizinszky, A. A. (2001). Yield Response of Bell Pepper Cultivars to Foliar-Applied‘Atonik’ Biostimulant. Bradenton: Horticultural Sciences Department, FloridaCooperative Extension Service, Institute of Food and Agricultural Sciences,University of Florida, HS819.

Czeczko, R., and Mikos-Bielak, M. (2004). Effects of Asahi bio-stimulator applica-tion in the cultivation of different vegetable species. Annales UMCS Sec. E 59,1073–1079.

Davies, P. J. (1987). Plant Hormones and their Role in Plant Growth andDevelopment. Dordrecht: Martinus Nijhoff Publishers. doi: 10.1007/978-94-009-3585-3

Djanaguiraman, M., Devi, D. D., Shanker, A. K., Sheeba, J. A., and Bangarusamy, U.(2004b). The role of nitrophenol on delaying abscission of tomato flowers andfruit. Food Agric. Environ. 2, 183–186.

Djanaguiraman, M., Devi, D. D., Sheeba, J. A., Bangarusamy, U., and Babu, R.C. H. (2004a). Effect of oxidative stress on abscission of tomato fruits and itsregulation by nitrophenols. Trop. Agric. Res. 16, 25–36.

Djanaguiraman, M., Sheeba, J. A., Devi, D. D., and Bangarusamy, U. (2005a). Effectof Atonik seed treatment on seedling physiology of cotton and tomato. J. Biol.Sci. 5, 163–169. doi: 10.3923/jbs.2005.163.169

Djanaguiraman, M., Sheeba, J. A., Devi, D. D., and Bangarusamy, U. (2005b).Response of cotton to Atonik and TIBA for growth, enzymes and yield. J. Biol.Sci. 5, 158–162. doi: 10.3923/jbs.2005.158.162

Djanaguiraman, M., Sheeba, J. A., Devi, D. D., and Bangarusamy, U. (2009).Cotton leaf senescence can be delayed by nitrophenolate spray throughenhanced antioxidant defence system. J. Agron. Crop Sci. 195, 213–224. doi:10.1111/j.1439-037X.2009.00360.x

Dobromilska, R., and Gubarewicz, K. (2008). “Influence of Bio-algeen S-90 on theyield and quality of small-sized tomato,” in Monographs Series: Biostimulators inModern Agriculture: Solanaceous Crops, ed Z. T. Dabrowski (Warsaw: EditorialHouse Wies Jutra), 7–12.





Gajc-Wolska, J., Kowalczyk, K., Nowecka, M., Mazur, K., and Metera, A. (2012).Effect of organic-mineral fertilizers on the field and quality of endive (Cichoriumendivia L.). Acta Sci Pol. Hortorum Cultus 11, 189–200.

Gajc-Wolska, J., Łyszkowska, M., and Zielony, T. (2010). The influence of graft-ing and biostimulators on the yield and fruit quality of greenhouse tomato cv.(Lycopersicon esculentum Mill.) grown in the field. Veget. Crops Res. Bull. 72,63–70. doi: 10.2478/v10032-010-0006-y

Gajc-Wolska, J., Radzanowska, J., and Łyszkowska, M. (2009). The influence ofgrafting and biostimulators on physical and sensorial traits of greenhousetomato fruit. (Lycopersicum esculentum Mill.) in field production. Acta Sci. Pol.Hortorum. Cultus 8, 37–43.

Gajewski, M., Gos, K., and Bobruk, J. (2008). “The influence of Goëmar Goteobiostimulator on yield and quality of two Chinese cabbage cultivars,” inMonographs Series: Biostimulators in Modern Agriculture: Vegetable Crops, ed Z.T. Dabrowski (Editorial House Wies Jutra), 21–27.

Gawlik, A., and Gołebiowska, D. (2008). “The influence of humic acids ongrowth of ‘Ramrod’ pea (Pisum sativum L.) plants,” in Book of Abstracts of theConference: Biostimulators in Modern Agriculture 7-8.02 (Warsaw).

Gawronska, H., Przybysz, A., Szalacha, E., and Słowinski, A. (2008). “Physiologicaland molecular mode of action of Asahi SL biostymulator under optima andstress conditions,” in Monographs Series: Biostimulators in Modern Agriculture:General Aspects, ed H. Gawronska (Editorial House Wies Jutra), 54–76.

Górnik, K., and Grzesik, M. (2002). Effect of Asahi SL on China aster ‘Aleksandra’seed yield, germination and some metabolic events. Acta Physiol. Plant 24,379–383. doi: 10.1007/s11738-002-0033-5

Górnik, K., and Grzesik, M. (2005). China aster plant growth, seed yield and qualityas influenced by Asahi SL treatment. Folia Hortic. 17, 119–127.

Górnik, K., and Grzesik, M. (2008). “Improvement of rooting and developmentof grapevine cuttings by Asahi SL, Biochikol 020PC, Tytanit, Citrosept andBiosept,” in Monographs Series: Biostimulators in Modern Agriculture: FruitCrops, ed A. Sadowski (Warsaw: Editorial House Wieœ Jutra), 31–41.

Górnik, K., Grzesik, M., and Mika, A. (2007). Improvement of grapevines foot-ing and growth of plants under stress conditions by Asahi SL. Folia Hortic. 19,57–67.

Grajkowski, J., and Ochmian, I. (2007). Influence of three biostimulants on yieldingand fruit quality of three primocane raspberry cultivars. Acta Sci. Pol. HortorumCultus 6, 29–36.

Gruszczyk, M., and Berbec, S. (2004). The effect of foliar application of some prepa-rations on yield and quality of feverfew (Chrysanthemum parthenium L.) rowmaterial. Ann. UMCS Sec. E 59, 755–759.

Gulluoglu, L., Arioglu, H., and Arslan, M. (2006). Effects of some plant growthregulators and nutrient complexes on above-ground biomass and seed yieldof soybean grown under heat-stressed environment. J. Agron. 5, 126–130. doi:10.3923/ja.2006.126.130

Harasimowicz-Hermann, G., and Czyz, K. (2008). “Effect of Asahi SL on the ini-tial development of willow cuttings at varied soil moisture,” in MonographsSeries: Biostimulators in Modern Agriculture: Ornament and Special Plants, edA. Łukaszewska (Warsaw: Editorial House Wies Jutra), 40–46.

Iturbe-Ormaetxe, I., Escuredo, P. R., Arrese-Igor, C., and Becana, M. (1998).Oxidative damage in pea exposed to water deficit or paraquat. Plan Physiol. 116,173–181. doi: 10.1104/pp.116.1.173

Khan, W., Rayirath, U. P., Subramanian, S., Jithesh, M. N., Rayorath, P., Hodges, D.M., et al. (2009). Seaweed extracts as biostimulants of plant growth and devel-opment. J. Plant Growth Regul. 4, 386–399. doi: 10.1007/s00344-009-9103-x

Koizumi, S., Maruyama, A., and Fulio, T. (1990). Purification on characterisationof ascorbic acid phosphorylating enzyme from Pseudomonas azotocolligans.Agric. Biol. Chem. 54, 3235–3239. doi: 10.1271/bbb1961.54.3235

Kołodziej, B. (2004). Wpływ Atoniku oraz nawozenia dolistnego na plonowaniei jakosc surowca zen-szenia amerykanskiego (Panax quinquefolium L.). Ann.UMCS Sec. E 59, 157–162.

Kołodziej, B. (2008). The effect of plantation establishment method and Atonikapplication in goldenrod (Solidago virgaurea L. ssp. virgaurea) cultivation. ActaSci. Pol Hortorum Cultus 7, 33–39.

Kositorna, J., and Smolinski, M. (2008). “Asahi SL biostimulator in protectionof sugar beet from herbicide stress,” in Monographs Series: Biostimulators inModern Agriculture: Field Crops, ed Z. T. Dabrowski (Warsaw: Editorial HouseWies Jutra), 41–49.

Kossak, K., and Dyki, B. (2008). “Effects of biostimulators on culture of AlboneyF1 greenhouse tomato,” in Monographs Series: Biostimulators in Modern

Agriculture: Solanaceous Crops, ed Z. T. Dabrowski (Warsaw: Editorial HouseWies Jutra), 13–20.

Kowalczyk, K., and Zielony, T. (2008). “Effect of Goteo treatment on yield and fruitquality of tomato grown on rockwool,” in Monographs Series: Biostimulators inModern Agriculture: Solanaceous Crops, ed Z. T. Dabrowski (Warsaw: EditorialHouse Wies Jutra), 21–26.

Kowalczyk, K., Zielony, T., and Gajewski, M. (2008). “Effect of Aminoplant andAsahi SL on yield and quality of lettuce grown on rockwool,” in MonographsSeries: Biostimulators in Modern Agriculture: Vegetable Crops, ed Z. T. Dabrowski(Warsaw: Editorial House Wies Jutra), 35–43.

Kozak, M., Malarz, W., Serafin-Andrzejewska, M., and Kotecki, A. (2008a). “Theeffect of sowing rate and Asahi SL biostimulator on soybean growth and yield,”in Monographs Series: Biostimulators in Modern Agriculture: Field Crops, ed Z. T.Dabrowski (Warsaw: Editorial House Wies Jutra), 77–84.

Kozak, M., Malarz, W., Serafin-Andrzejewska, M., and Kotecki, A. (2008b).“The effect of different sowing rate and Asahi SL treatment on soybeansowing value,” in Monographs Series: Biostimulators in Modern Agriculture:Field Crops, ed Z. T. Dabrowski (Warsaw: Editorial House Wies Jutra),85–91.

Krajewska, J., and Latkowska, M. J. (2008). “Effects of biostimulants Asahi SL andSiapton 10L on the growth of Bergenia cordifolia ((Haw.) Sternb.) ‘Rotblum’and Hosta sp. (Tratt.) ‘Sum and Substance’ and ‘Minuteman’,” in MonographsSeries: Biostimulators in Modern Agriculture: Ornament and Special Plants, ed A.Łukaszewska (Warsaw: Editorial House Wies Jutra), 33–39.

Krawczyk, R., and Skoczynski, J. (2008). “Winter survival and yield of oilseed rapedepending on sowing date and application of micronutrient preparation Route®acting as a growth stimulator,” in Monographs Series: Biostimulators in ModernAgriculture: Field Crops, ed Z. T. Dabrowski (Warsaw: Editorial House WiesJutra), 33–40.

Krawiec, P. (2008). “Effects of biostimulators on growth, cropping and fruit qual-ity of chokeberry,” in Monographs Series: Biostimulators in Modern Agriculture:Fruit Crops, ed A. Sadowski (Warsaw: Editorial House Wies Jutra), 42–48.

Krok, K., and Wieniarska, J. (2008). “Effect of Goëmar BM 86 application ondevelopment and quality of primocane raspberry fruits,” in Monographs Series:Biostimulators in Modern Agriculture: Fruit Crops, ed A. Sadowski (Warsaw:Editorial House Wies Jutra), 49–59.

Ksiezak, J. (2008). “Effect of biostimulator Asahi SL on yield of maize grain,” inMonographs Series: Biostimulators in Modern Agriculture: Field Crops, ed Z. T.Dabrowski (Warsaw: Editorial House Wies Jutra), 60–65.

Libbenga, K. R., and Mennes, A. M. (1987). “Hormone binding and its rolein hormone action,” in Plant Hormones and their Role in Plant Growth andDevelopment, ed P.J. Davies (Dordrecht: Martinus Nijhoff Publishers), 194–221.doi: 10.1007/978-94-009-3585-3_11

Łyszkowska, M., Gajc-Wolska, J., and Kubis, K. (2008). “The influence of bios-timulator on yield and quality of leaf and iceberg lettuce – grown underfield conditions,” in Monographs Series: Biostimulators in Modern Agriculture:Vegetable Crops, ed Z. T. Dabrowski (Warsaw: Editorial House Wies Jutra),28–34.

Maciejewski, T., Michalski, T., Bartos-Spychała, M., and Cieslicki, W. (2008).“Effect of the application of the biostimulator Asahi SL on the yield ofpotato tubers and their quality,” in Monograph Series: Biostimulators in ModernAgriculture: Solanaceous Crops, ed Z. T. Dabrowski (Warsaw: Editorial HouseWies Jutra), 52–60.

Malarz, W., Kozak, M., and Kotecki, A. (2008). “The use of Asahi SL biostimu-lator in spring rape growing,” in Monographs Series: Biostimulators in ModernAgriculture: Field Crops, ed Z. T. Dabrowski (Warsaw: Editorial House WiesJutra), 25–32.

Matysiak, K., and Kaczmarek, S. (2008). “Potential advantages of Kelpak bioregula-tor applied to some field crops,” in Monographs Series: Biostimulators in ModernAgriculture: Field Crops, ed Z. T. Dabrowski (Warsaw: Editorial House WiesJutra), 99–108.

Michalski, T., Bartos-Sychała, M., Maciejewski, T., and Jarosz, A. (2008). “Effect ofbiostymulator Asahi SL on cropping of maize grown for grain,” in MonographsSeries: Biostimulators in Modern Agriculture: Field Crops, ed Z. T. Dabrowski(Warsaw: Editorial House Wies Jutra), 66–76.

Miranda-Stalder, S. H., Gloria, B. A., and Castro, P. R. C. (1990). Effect of growthregulators on morphological characteristics and productivity of strawberry“Sequoia’. An Esc Super Agric, Luiz de Queiroz 47, 317–334. doi: 10.1590/S0071-12761990000200005






Nair, P., Kandasamy, S., Zhang, J., Ji, X., Kirby, C., Benkel, B., et al. (2012).Transcriptional and metabolomic analysis of Ascophyllum nodosum medi-ated freezing tolerance in Arabidopsis thaliana. BMC Genomics 13:643. doi:10.1186/1471-2164-13-643

Ochmian, I., Grajkowski, J., and Skupien, K. (2008). “Influence of three biostim-ulators on growth, yield and fruit chemical composition of ‘Polka’ raspberry,”in Monographs Series: Biostimulators in Modern Agriculture: Fruit Crops, ed A.Sadowski (Warsaw: Editorial House Wies Jutra), 68–77.

Oosterhuis, D. (2008). “Atonik™ biostimulators for increased nitrogen, protein andyield of cotton,” in Book of Abstracts of the Conference Biostimulators in ModernAgriculture 7-8.02 (Warsaw), 18.

Oosterhuis, D., and Robertson, W. C. (2000). “The use plant growth regulatorsand other additives in cotton production,”in Arkansas Agricultural ExperimentStation Special Report 198, Proceedings of the 2000 Cotton Meeting (Fayetteville,AR), 22–32.

Panajotov, N. D., Jevtic, S., and Lazic, B. (1997). Sweet pepper response to theapplication of the plant growth regulator Atonic. Acta Hortic. 462, 197–202.

Przybysz, A., Janowiak, F., Słowinski, A., and Gawronska, H. (2010). Protectiverole of Asahi SL against drought stress. Zeszyty Problemowe Postepów NaukRolniczych PAN 545, 199–223.

Roberts, D. M., and Harmon, A. C. (1992). Calcium-modulated proteins: targetsof intracellular calcium signals in higher plants. Annu. Rev. Plant Phys. 43,375–414. doi: 10.1146/annurev.pp.43.060192.002111

Sas-Paszt, L., Zurawicz, E., Masny, A., Filipczak, J., Pluta, S., Lewandowski, M., et al.(2008). “The use of biostimulators in small fruit growing,” in Monograph Series:Biostimulators in Modern Agriculture: Field Crops, ed Z. T. Dabrowski (Warsaw:Editorial House Wies Jutra), 76–90.

Sawicka, B., and Mikos-Bielak, M. (2008). “Modification of potato tuber chemicalcomposition by applications of the Asahi SL biostimulator,” in Monograph series:Biostimulators in Modern Agriculture: Solanaceous Crops, ed Z. T. Dabrowski(Warsaw: Editorial House Wies Jutra), 61–67.

Schroeder, J. I., Allen, G. J., Hugouvieux, V., Kwak, J. M., and Waner, D. (2001).Guard cell signal transduction. Annu. Rev. Plant Phys. 52, 627–658. doi:10.1146/annurev.arplant.52.1.627

Sharma, R., Sharma, B., and Singh, G. (1984). Phenols as regulators of nitratereductase activity in Cicer arietiman. �YTON 44, 185–188.

Shinozaki, K., and Yamaguchi-Schinozaki, K. (2007). Gene networks involvedin drought stress response and tolerance. J. Exp. Bot. 58, 221–227. doi:10.1093/jxb/erl164

Siedlecka, A., and Krupa, Z. (2002). Simple method of Arabidopsis thaliana cul-tivation in liquid nutrient medium. Acta Physiol. Plant 24, 163–166. doi:10.1007/s11738-002-0007-7

Stepowska, A. (2008a). “Biostimulators in sweet pepper cultivation under covers,”in Monographs Series: Biostimulators in Modern Agriculture: Solanaceous Crops,ed Z. T. Dabrowski (Warsaw: Editorial House Wies Jutra), 36–44.

Stepowska, A. (2008b). “Effect of GA 142 (Goëmar Goteo) and (Goëmar BM 86)extracts on sweet pepper yield in non-heated tunnels,” in Monographs Series:Biostimulators in Modern Agriculture: Solanaceous Crops, ed Z. T. Dabrowski(Warsaw: Editorial House Wies Jutra), 45–51.

Stutte, C. A., and Clark, T. H. (1990). Radiolabeled Studies of Atonik in Cottonusing HPLC. Arysta LifeScience Report. Fayetteville, AR: Altheimer Laboratory,University of Arkansas.

Stutte, C. H. A., Urwiler, M. J., and Clark, T. H. (1987). Laboratory and FieldEvaluation of Atonik on Cotton. Arysta LifeScience Report. Fayetteville, AR:University of Arkansas.

Taiz, L., and Zeiger, E. (2002). Plant Physiology. Sunderland: Sinauer Associates,Inc., Publishers.

Wilson, G. F., and Kaczmarek, L. K. (1993). Mode-switching of voltage-gated cationchannel is mediated by a protein kinase A-regulated tyrosine phosphatase.Nature 366, 433–438. doi: 10.1038/366433a0

Wróbel, J., and Wozniak, A. (2008). “The effect of Atonik plant growth stimulatoron physiological indicators of the basket willow (Salix viminalis L.) cultivated inanthropogenic soil,” in Monographs Series: Biostimulators in Modern Agriculture:Ornament and Special Plants, ed A. Łukaszewska (Warsaw: Editorial House WiesJutra), 47–55.

Wrochna, M., Łata, B., Borkowska, B., and Gawronska, H. (2008). “The effect AsahiSL of biostimulators on ornament amaranth (Amaranthus sp.) plants exposedto salinity in growing medium,” in Monographs Series: Biostimulators in ModernAgriculture: Ornament and Special Plants, ed A. Łukaszewska (Warsaw: EditorialHouse Wies Jutra), 15–32.

Wrona, D., and Misiura, M. (2008). “Effect of Goëmar BM 86 on yieldand quality,” in Monographs Series: Biostimulators in Modern Agriculture:Fruit Crops, ed A. Sadowski (Warsaw: Editorial House Wies Jutra),91–96.

Yamaki, T., Nakasawa, K., Nakamura, K., Terakawa, H., and Hayashi, T. (1953). Aplant physiological study of Atonik (Advance Report). Agric. Hortic. 28, 1–3.

Zhao, D., and Oosterhuis, D. (1997). Physiological response of growthchamber-grown cotton plants to the plant growth regulator PGR-IV underwater-deficit stress. Environ. Exp. Bot. 38, 7–14. doi: 10.1016/S0098-8472(97)00002-6

Conflict of Interest Statement: The authors declare that the research was con-ducted in the absence of any commercial or financial relationships that could beconstrued as a potential conflict of interest.

Received: 16 July 2014; accepted: 27 November 2014; published online: 16 December2014.Citation: Przybysz A, Gawronska H and Gajc-Wolska J (2014) Biological mode ofaction of a nitrophenolates-based biostimulant: case study. Front. Plant Sci. 5:713.doi: 10.3389/fpls.2014.00713This article was submitted to Crop Science and Horticulture, a section of the journalFrontiers in Plant Science.Copyright © 2014 Przybysz, Gawronska and Gajc-Wolska. This is an open-accessarticle distributed under the terms of the Creative Commons Attribution License(CC BY). The use, distribution or reproduction in other forums is permitted, providedthe original author(s) or licensor are credited and that the original publication in thisjournal is cited, in accordance with accepted academic practice. No use, distribution orreproduction is permitted which does not comply with these terms.


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Biological mode of action of a nitrophenolates-based ... · Przybysz et al. Mode of action of a nitrophenolates-based biostimulant (0.1%), and water. Atonik has been used successfully

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